Transcription of major histocompatibility complex (MHC) class I genes is regulated by both tissue-specific (basal) and hormone/cytokine (activated) mechanisms. Although promoter-proximal regulatory elements have been characterized extensively, the roles of the core promoter and downstream elements in mediating regulation have been largely undefined. Basal and activated transcriptions of an MHC class I gene target distinct core promoter domains, nucleate distinct transcription initiation complexes and initiate at distinct sites within the promoter. Basal and activated transcription pathways recruit distinct transcription factor complexes to the core promoter elements and target distinct transcription initiation sites. Basal transcription is completely dependent upon the general transcription factor TAF1 whereas activated transcription is TAF1 independent. To further characterize regulation of class I gene expression, we have undertaken to characterize core promoter elements in vivo and to identify novel downstream promoter elements. To understand these mechanisms in more detail, we have characterized elements within the core promoter of the MHC class I gene. The minimal class I core promoter has been localized to a segment between -50 bp and +14 bp. Within this segment are sequences similar to canonical TATA and Inr promoter elements and an Sp1 binding site. In past studies, we have reported that no single element is necessary for expression of a reporter in transient transfection assays. However, we have now examined the dependence of transcription on each of the core promoter elements in the context of the native gene in transgenic mice. Remarkably, in parallel with the transfection data, we find that no single element is necessary for in vivo expression: all of the core promoter mutations result in actively expressed MHC class I genes. Rather, the elements regulate either promoter strength or tissue tissue specific patterns of expression and response to cytokines is markedly affected by the different mutations.We have also found that the downstream region of the MHC class I promoter region, between +1 and +32 bp, contains three novel regulatory elements. One of the elements,E+4, functions to increase transcription. The other two elements, DPE-L1 and DPE-L2 have sequence homology with previously characterized DPE elements, but are mechanistically and functionally distinct from other described DPE elements. Under constitutive conditions, the two DPE-L's act in concert to up-regulate promoter activity, preferentially increasing the use of TSS located at the 5' end of the cluster of multiple start sites. These elements promote constitutive, TAF1-dependent transcription. However, under activated TAF1-independent conditions, only one of the element functions independently as an enhancer. Thus, the downstream regulatory elements associated with the class I promoter function to fine-tune and integrate both intracellular and extracellular signaling pathways to ensure the appropriate level of MHC class I transcription to maintain immune homeostasis. These novel downstream elements are functionally and mechanistically distinct from previously described downstream elements in mammalian promoters. Importantly, we have now identified a second promoter element within the core promoter region. This novel promoter element alone is capable of supporting transcription of a reporter gene in the presence or a heterologous enhancer. Furthermore, it is capable of supporting CIITA-activated transcription of a reporter gene. Most significantly, this novel promoter drives the expression of the native MHC class I gene both in stably transfected L cells and in transgenic mice. Thus, this promoter element, like the first, is capable of supporting both constitutive and activated transcription in vitro and in vivo. The roles of the two distinct promoters in regulating MHC class I transcription is an active area of investigation.